Abstract B05: Intestinal microbiota mediates pancreatic carcinogenesis through modulation of tumor infiltrating natural killer cells

Abstract

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related death in the United States. Our group and others have demonstrated that the intestinal microbiota accelerates pancreatic carcinogenesis. The relationship of intestinal bacteria, immune response, and PDAC development is unclear. To that end, we investigated the role of intestinal bacterial soluble factors in modulating the immune environment of PDAC and its progression. Methods: Mice intestinal microbiota was depleted with wide-spectrum antibiotics. The human PDAC cell line, L3.6pl, was heterotopically implanted into Rag1-/- mice, while the syngeneic murine PDAC cell line, Pan02, was orthotopically implanted into the pancreas of C57Bl/6 mice. Tumor and pancreas infiltrated natural killer (NK) cells were quantitated by flow cytometry. In vivo NK cell depletion was attained by intraperitoneal injection of anti-Asialo-GM1 antibody twice weekly. Germ-free (GF) and conventionally housed Rag1-/- mice stool was cultured and bacteria-free supernatant extracted. The ability of these bacteria-free supernatants to regulate NK-92mi cell cytotoxicity and migration was tested in vitro by flow cytometry and a Transwell assay, respectively. Results: Compared to microbiota-intact Rag1-/- mice, microbiota depletion yielded 74% smaller tumors (p<0.05) with a 1.5-fold increase in PDAC infiltrating NK cells (p<0.001). Confirmation by immunohistochemistry demonstrated a 6.5-fold increase in NK cell tumor infiltration in microbiota-depleted mice (p<0.0001). Notably, a 3-fold increase of intrapancreatic NK cells in GF Rag1-/- mice versus conventionally housed mice was noted, suggesting that bacteria regulate immune cell trafficking. Antibody-mediated NK cell depletion with concomitant microbiota depletion increased PDAC tumor growth, negating the antitumor phenotype of microbiota depletion alone in both immunodeficient Rag1-/- (2.8-fold increase; p<0.05) and immunocompetent C57Bl/6 mice (2.4-fold increase; p<0.05). Quantitative PCR revealed 9-fold higher IFN-gamma gene expression in PDAC xenografts of microbiota-depleted mice versus microbiota-intact (p<0.05). Cell-free stool bacteria culture supernatant from conventionally housed Rag1-/- mice, but not from GF mice, inhibited NK-92mi cell migration by 26% (p<0.05) and cytotoxicity against L3.6pl cells by 51% (p<0.01). Compared to NK-92mi cells exposed to cultured stool supernatant from GF Rag1-/- mice, SPF cultured stool supernatant resulted in decreased gene expression associated with activation/recruitment of NK cells including FASLG, CCL18, IL-13, CXCR2, CCL4, and increased expression associated with inhibition of NK cell activity including CCR1 and IL-6. Conclusion: These findings suggest that intestinal bacteria modulate PDAC development through suppression of NK cell recruitment and activation, a phenomenon potentially mediated by yet to be identified small molecules. Citation Format: Qin Yu, Mark Beveridge, Ryan Thomas, Christian Jobin. Intestinal microbiota mediates pancreatic carcinogenesis through modulation of tumor infiltrating natural killer cells [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B05.